Toxin genes and methods for their use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a delta-endotoxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated delta-endotoxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:61-121 and 133-141, or the nucleotide sequence set forth in SEQ ID NO:1-60, 124-132, and 142-283, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/491,396, filed Jun. 25, 2009, which claims the benefit of U.S.Provisional Application Ser. No. 61/075,719, filed Jun. 25, 2008, andU.S. Provisional Application Ser. No. 61/158,137, filed Mar. 6, 2009,the contents of which are herein incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA057US02SEQLIST.txt”, created on May 14, 2013, and having a size of1,075 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Dipteran, and Coleopteran larvae. These proteins also have shownactivity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, andAcari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, Cry1C2, etc.

A new nomenclature was recently described for the Cry genes based uponamino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

The crystal protein does not exhibit insecticidal activity until it hasbeen ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Höfte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

The intensive use of B. thuringiensis-based insecticides has alreadygiven rise to resistance in field populations of the diamondback moth,Plutella xylostella (Ferre and Van Rie (2002) Annu. Rev. Entomol.47:501-533). The most common mechanism of resistance is the reduction ofbinding of the toxin to its specific midgut receptor(s). This may alsoconfer cross-resistance to other toxins that share the same receptor(Ferre and Van Rie (2002)).

SUMMARY OF INVENTION

Compositions and methods for conferring pest resistance to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for delta-endotoxinpolypeptides, vectors comprising those nucleic acid molecules, and hostcells comprising the vectors. Compositions also include the polypeptidesequences of the endotoxin, and antibodies to those polypeptides. Thenucleotide sequences can be used in DNA constructs or expressioncassettes for transformation and expression in organisms, includingmicroorganisms and plants. The nucleotide or amino acid sequences may besynthetic sequences that have been designed for expression in anorganism including, but not limited to, a microorganism or a plant.Compositions also comprise transformed bacteria, plants, plant cells,tissues, and seeds.

In particular, isolated nucleic acid molecules corresponding todelta-endotoxin nucleic acid sequences are provided. Additionally, aminoacid sequences corresponding to the polynucleotides are encompassed. Inparticular, the present invention provides for an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence shown in any of SEQ ID NO:61-121 and 133-141, or a nucleotidesequence set forth in any of SEQ ID NO:1-60 and 124-132, as well asvariants and fragments thereof. Nucleotide sequences that arecomplementary to a nucleotide sequence of the invention, or thathybridize to a sequence of the invention are also encompassed.

The compositions and methods of the invention are useful for theproduction of organisms with pesticide resistance, specifically bacteriaand plants. These organisms and compositions derived from them aredesirable for agricultural purposes. The compositions of the inventionare also useful for generating altered or improved delta-endotoxinproteins that have pesticidal activity, or for detecting the presence ofdelta-endotoxin proteins or nucleic acids in products or organisms.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance in organisms, particularly plants or plantcells. The methods involve transforming organisms with a nucleotidesequence encoding a delta-endotoxin protein of the invention. Inparticular, the nucleotide sequences of the invention are useful forpreparing plants and microorganisms that possess pesticidal activity.Thus, transformed bacteria, plants, plant cells, plant tissues and seedsare provided. Compositions are delta-endotoxin nucleic acids andproteins of Bacillus thuringiensis. The sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of otherdelta-endotoxin genes, and for the generation of altered pesticidalproteins by methods known in the art, such as domain swapping or DNAshuffling. The proteins find use in controlling or killing lepidopteran,coleopteran, and nematode pest populations, and for producingcompositions with pesticidal activity.

By “delta-endotoxin” is intended a toxin from Bacillus thuringiensisthat has toxic activity against one or more pests, including, but notlimited to, members of the Lepidoptera, Diptera, and Coleoptera ordersor members of the Nematoda phylum, or a protein that has homology tosuch a protein. In some cases, delta-endotoxin proteins have beenisolated from other organisms, including Clostridium bifermentans andPaenibacillus popilliae. Delta-endotoxin proteins include amino acidsequences deduced from the full-length nucleotide sequences disclosedherein, and amino acid sequences that are shorter than the full-lengthsequences, either due to the use of an alternate downstream start site,or due to processing that produces a shorter protein having pesticidalactivity. Processing may occur in the organism the protein is expressedin, or in the pest after ingestion of the protein.

Delta-endotoxins include proteins identified as cry1 through cry43, cyt1and cyt2, and Cyt-like toxin. There are currently over 250 known speciesof delta-endotoxins with a wide range of specificities and toxicities.For an expansive list see Crickmore et al. (1998), Microbiol. Mol. Biol.Rev. 62:807-813, and for regular updates see Crickmore et al. (2003)“Bacillus thuringiensis toxin nomenclature,” atwww.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.

Provided herein are novel isolated nucleotide sequences that conferpesticidal activity. Also provided are the amino acid sequences of thedelta-endotoxin proteins. The protein resulting from translation of thisgene allows cells to control or kill pests that ingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding delta-endotoxinproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify delta-endotoxin encoding nucleic acids. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolateddelta-endotoxin encoding nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A delta-endotoxinprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of non-delta-endotoxin protein (also referred to herein as a“contaminating protein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:1-60 and 124-132, andvariants, fragments, and complements thereof. By “complement” isintended a nucleotide sequence that is sufficiently complementary to agiven nucleotide sequence such that it can hybridize to the givennucleotide sequence to thereby form a stable duplex. The correspondingamino acid sequence for the delta-endotoxin protein encoded by thisnucleotide sequence are set forth in SEQ ID NO:61-121 and 133-141.

Nucleic acid molecules that are fragments of these delta-endotoxinencoding nucleotide sequences are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a delta-endotoxin protein. A fragment of a nucleotidesequence may encode a biologically active portion of a delta-endotoxinprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a delta-endotoxin nucleotide sequencecomprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150,2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750,2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350contiguous nucleotides, or up to the number of nucleotides present in afull-length delta-endotoxin encoding nucleotide sequence disclosedherein depending upon the intended use. By “contiguous” nucleotides isintended nucleotide residues that are immediately adjacent to oneanother. Fragments of the nucleotide sequences of the present inventionwill encode protein fragments that retain the biological activity of thedelta-endotoxin protein and, hence, retain pesticidal activity. By“retains activity” is intended that the fragment will have at leastabout 30%, at least about 50%, at least about 70%, 80%, 90%, 95% orhigher of the pesticidal activity of the delta-endotoxin protein.Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485;Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J.of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety.

A fragment of a delta-endotoxin encoding nucleotide sequence thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100 contiguous amino acids, or up to the total number ofamino acids present in a full-length delta-endotoxin protein of theinvention.

Preferred delta-endotoxin proteins of the present invention are encodedby a nucleotide sequence sufficiently identical to the nucleotidesequence of SEQ ID NO:1-60 and 124-132. By “sufficiently identical” isintended an amino acid or nucleotide sequence that has at least about60% or 65% sequence identity, about 70% or 75% sequence identity, about80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of one ofSEQ ID NO:1-60 and 124-132, or across the entirety of one of SEQ IDNO:61-121 and 133-141). The percent identity between two sequences canbe determined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous todelta-endotoxin-like nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous todelta-endotoxin protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389. Alternatively, PSI-Blast can be used to perform an iteratedsearch that detects distant relationships between molecules. SeeAltschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., BLASTX and BLASTN) can be used. Alignment may also be performedmanually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The invention also encompasses variant nucleic acid molecules.“Variants” of the delta-endotoxin encoding nucleotide sequences includethose sequences that encode the delta-endotoxin proteins disclosedherein but that differ conservatively because of the degeneracy of thegenetic code as well as those that are sufficiently identical asdiscussed above. Naturally occurring allelic variants can be identifiedwith the use of well-known molecular biology techniques, such aspolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the delta-endotoxinproteins disclosed in the present invention as discussed below. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, retaining pesticidal activity. By “retainsactivity” is intended that the variant will have at least about 30%, atleast about 50%, at least about 70%, or at least about 80% of thepesticidal activity of the native protein. Methods for measuringpesticidal activity are well known in the art. See, for example, Czaplaand Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988)Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology78:290-293; and U.S. Pat. No. 5,743,477, all of which are hereinincorporated by reference in their entirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodeddelta-endotoxin proteins, without altering the biological activity ofthe proteins. Thus, variant isolated nucleic acid molecules can becreated by introducing one or more nucleotide substitutions, additions,or deletions into the corresponding nucleotide sequence disclosedherein, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Such variant nucleotide sequences are alsoencompassed by the present invention.

For example, conservative amino acid substitutions may be made at one ormore predicted, nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of a delta-endotoxin protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of the amino acidsequences of the present invention and known delta-endotoxin sequences.Examples of residues that are conserved but that may allow conservativeamino acid substitutions and still retain activity include, for example,residues that have only conservative substitutions between all proteinscontained in an alignment of the amino acid sequences of the presentinvention and known delta-endotoxin sequences. However, one of skill inthe art would understand that functional variants may have minorconserved or nonconserved alterations in the conserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer delta-endotoxin activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingdelta-endotoxin sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the delta-endotoxin nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known delta-endotoxin-encodingnucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in the nucleotidesequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,or 400 consecutive nucleotides of delta-endotoxin encoding nucleotidesequence of the invention or a fragment or variant thereof. Methods forthe preparation of probes for hybridization are generally known in theart and are disclosed in Sambrook and Russell, 2001, supra hereinincorporated by reference.

For example, an entire delta-endotoxin sequence disclosed herein, or oneor more portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding delta-endotoxin-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, or at least about 20nucleotides in length. Such probes may be used to amplify correspondingdelta-endotoxin sequences from a chosen organism by PCR. This techniquemay be used to isolate additional coding sequences from a desiredorganism or as a diagnostic assay to determine the presence of codingsequences in an organism. Hybridization techniques include hybridizationscreening of plated DNA libraries (either plaques or colonies; see, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Delta-endotoxin proteins are also encompassed within the presentinvention. By “delta-endotoxin protein” is intended a protein having theamino acid sequence set forth in SEQ ID NO:61-121 and 133-141.Fragments, biologically active portions, and variants thereof are alsoprovided, and may be used to practice the methods of the presentinvention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in any of SEQ ID NO:61-121 and 133-141 andthat exhibit pesticidal activity. A biologically active portion of adelta-endotoxin protein can be a polypeptide that is, for example, 10,25, 50, 100 or more amino acids in length. Such biologically activeportions can be prepared by recombinant techniques and evaluated forpesticidal activity. Methods for measuring pesticidal activity are wellknown in the art. See, for example, Czapla and Lang (1990) J. Econ.Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206;Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S.Pat. No. 5,743,477, all of which are herein incorporated by reference intheir entirety. As used here, a fragment comprises at least 8 contiguousamino acids of SEQ ID NO:61-121 and 133-141. The invention encompassesother fragments, however, such as any fragment in the protein greaterthan about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, or 1300 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of any of SEQ ID NO:61-121 and 133-141.Variants also include polypeptides encoded by a nucleic acid moleculethat hybridizes to the nucleic acid molecule of SEQ ID NO:1-60 and124-132, or a complement thereof, under stringent conditions. Variantsinclude polypeptides that differ in amino acid sequence due tomutagenesis. Variant proteins encompassed by the present invention arebiologically active, that is they continue to possess the desiredbiological activity of the native protein, that is, retaining pesticidalactivity. Methods for measuring pesticidal activity are well known inthe art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. However, bacteria such asBacillus sp. also recognize the codon GTG as a start codon, and proteinsthat initiate translation at GTG codons contain a methionine at thefirst amino acid. Furthermore, it is not often determined a priori whichof these codons are used naturally in the bacterium. Thus, it isunderstood that use of one of the alternate methionine codons may alsolead to generation of delta-endotoxin proteins that encode pesticidalactivity. These delta-endotoxin proteins are encompassed in the presentinvention and may be used in the methods of the present invention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a delta-endotoxin may be alteredby various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a delta-endotoxin of the present invention. This proteinmay be altered in various ways including amino acid substitutions,deletions, truncations, and insertions of one or more amino acids of SEQID NO:61-121 and 133-141, including up to about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 100, about 105, about 110, about 115, about 120, about 125,about 130 or more amino acid substitutions, deletions or insertions.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a delta-endotoxin protein canbe prepared by mutations in the DNA. This may also be accomplished byone of several forms of mutagenesis and/or in directed evolution. Insome aspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of a delta-endotoxin to confer pesticidal activity may beimproved by the use of such techniques upon the compositions of thisinvention. For example, one may express a delta-endotoxin in host cellsthat exhibit high rates of base misincorporation during DNA replication,such as XL-1 Red (Stratagene). After propagation in such strains, onecan isolate the delta-endotoxin DNA (for example by preparing plasmidDNA, or by amplifying by PCR and cloning the resulting PCR fragment intoa vector), culture the delta-endotoxin mutations in a non-mutagenicstrain, and identify mutated delta-endotoxin genes with pesticidalactivity, for example by performing an assay to test for pesticidalactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests. Examples of mutations that result in increasedtoxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev.62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent delta-endotoxin protein coding regions can be used to create anew delta-endotoxin protein possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled between adelta-endotoxin gene of the invention and other known delta-endotoxingenes to obtain a new gene coding for a protein with an improvedproperty of interest, such as an increased insecticidal activity.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating altereddelta-endotoxin proteins. Domains II and III may be swapped betweendelta-endotoxin proteins, resulting in hybrid or chimeric toxins withimproved pesticidal activity or target spectrum. Methods for generatingrecombinant proteins and testing them for pesticidal activity are wellknown in the art (see, for example, Naimov et al. (2001) Appl. Environ.Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ.Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem.266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930;Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925).

Vectors

A delta-endotoxin sequence of the invention may be provided in anexpression cassette for expression in a plant of interest. By “plantexpression cassette” is intended a DNA construct that is capable ofresulting in the expression of a protein from an open reading frame in aplant cell. Typically these contain a promoter and a coding sequence.Often, such constructs will also contain a 3′ untranslated region. Suchconstructs may contain a “signal sequence” or “leader sequence” tofacilitate co-translational or post-translational transport of thepeptide to certain intracellular structures such as the chloroplast (orother plastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.By “leader sequence” is intended any sequence that when translated,results in an amino acid sequence sufficient to trigger co-translationaltransport of the peptide chain to a sub-cellular organelle. Thus, thisincludes leader sequences targeting transport and/or glycosylation bypassage into the endoplasmic reticulum, passage to vacuoles, plastidsincluding chloroplasts, mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the delta-endotoxin sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the delta-endotoxin is targeted to the chloroplastfor expression. In this manner, where the delta-endotoxin is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe delta-endotoxin to the chloroplasts. Such transit peptides are knownin the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol.Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550;Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al.(1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al.(1986) Science 233:478-481.

The delta-endotoxin gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The delta-endotoxin gene of the inventionmay be modified to obtain or enhance expression in plant cells.Typically a construct that expresses such a protein would contain apromoter to drive transcription of the gene, as well as a 3′untranslated region to allow transcription termination andpolyadenylation. The organization of such constructs is well known inthe art. In some instances, it may be useful to engineer the gene suchthat the resulting peptide is secreted, or otherwise targeted within theplant cell. For example, the gene can be engineered to contain a signalpeptide to facilitate transfer of the peptide to the endoplasmicreticulum. It may also be preferable to engineer the plant expressioncassette to contain an intron, such that mRNA processing of the intronis required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the delta-endotoxin are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the delta-endotoxin is then tested by hybridizing the filterto a radioactive probe derived from a delta-endotoxin, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thedelta-endotoxin gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thedelta-endotoxin protein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a delta-endotoxin that has pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a delta-endotoxin may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a delta-endotoxin may be tested for pesticidalactivity, and the plants showing optimal activity selected for furtherbreeding. Methods are available in the art to assay for pest activity.Generally, the protein is mixed and used in feeding assays. See, forexample Marrone et al. (1985) J. of Economic Entomology 78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape., etc.).

Use in Pest Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pesticide control orin engineering other organisms as pesticidal agents are known in theart. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene and protein may be usedfor protecting agricultural crops and products from pests. In one aspectof the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticide is produced by introducing adelta-endotoxin gene into a cellular host. Expression of thedelta-endotoxin gene results, directly or indirectly, in theintracellular production and maintenance of the pesticide. In one aspectof this invention, these cells are then treated under conditions thatprolong the activity of the toxin produced in the cell when the cell isapplied to the environment of target pest(s). The resulting productretains the toxicity of the toxin. These naturally encapsulatedpesticides may then be formulated in accordance with conventionaltechniques for application to the environment hosting a target pest,e.g., soil, water, and foliage of plants. See, for example EPA 0192319,and the references cited therein. Alternatively, one may formulate thecells expressing a gene of this invention such as to allow applicationof the resulting material as a pesticide.

Pesticidal Compositions

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, coleopteran, or nematode pests may be killed or reduced innumbers in a given area by the methods of the invention, or may beprophylactically applied to an environmental area to prevent infestationby a susceptible pest. Preferably the pest ingests, or is contactedwith, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrimidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylimidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachinidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseintroducing into a plant or plant cell a polynucleotide comprising apesticidal sequence disclosed herein. As defined herein, the “yield” ofthe plant refers to the quality and/or quantity of biomass produced bythe plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing thepesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a pesticidal protein disclosedherein. Expression of the pesticidal protein results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Discovery of a Novel Pesticidal Genes fromBacillus thuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 1 using the following steps:

-   -   Preparation of extrachromosomal DNA from the strain, which        includes plasmids that typically harbor delta-endotoxin genes    -   Mechanical shearing of extrachromosomal DNA to generate        size-distributed fragments    -   Cloning of ˜2 Kb to ˜10 Kb fragments of extrachromosomal DNA    -   Outgrowth of ˜1500 clones of the extrachromosomal DNA    -   Partial sequencing of the 1500 clones using primers specific to        the cloning vector (end reads)    -   Identification of putative toxin genes via homology analysis via        the MiDAS approach (as described in U.S. Patent Publication No.        20040014091, which is herein incorporated by reference in its        entirety)    -   Sequence finishing (walking) of clones containing fragments of        the putative toxin genes of interest

TABLE 1 List of novel genes isolated from Bacillus thuringiensis PercentAmino identity to Nucleotide Acid closest Closest SEQ ID SEQ ID sequencesequence Gene Strain NO: NO: in art in art axmi046¹ ATX13026 1 6154.30%  Cry4Aa1 axmi048² ATX13026 2 62 56.4% Cry4Ba1 axmi050³ ATX21049 363 17.9% Cry21Ba1 axmi051⁴ ATX21049 4 64 21.3% Cry35Ac1 axmi052⁵ATX21049 5 65 19.70%  Cry35Aa1 axmi053 ATX21049 6 66 21.9% Cry35Ac1axmi054 ATX21049 7 67 20.1% Cry35Ac1 axmi055 ATX12976 8 68 42.0%Cry32Ca1 axmi056 ATX12976 9 69 (homology BinA/BinB to N- terminus)axmi057 ATX13058 10 70 73.1% Cry32Da1 axmi058 ATX13058 11 71 26.8%Cry6Ba1 axmi059 ATX13058 12 72 56.0% Cry32Aa1 axmi060 ATX13058 13 7354.6% Cry32Aa1 axmi061 ATX13058 14 74 29.2% Cry45Aa1 axmi067 ATX12974 1575 36.3% Cry32Da1 axmi069 ATX12997 17 77 Cry32Ca1 Some N-terminalhomology axmi071 ATX12982 18 78 22.9% Cry21Ba1 axmi072 ATX12982 19 7916.4% Mtx2 axmi073 ATX16042 20 80 15.3% Mtx2 axmi074 ATX12993 21 8143.8% Cry21Ba1 axmi075 ATX12997 22 82 30.4% Cry32Da1 axmi087 ATX13030 2787 71.0% Cry8Aa1 axmi088 ATX13039 28 88 26.2% Cry21Ba1 axmi093 ATX1305831 91 56.1% Cry32Aa1 ¹Potential co-activity when expressed or pairedwith another toxin such as Axmi0014 or Axmi008 ²Potential co-activitywhen expressed or paired with another toxin such as Axmi0014 or Axmi009³An N-terminal domain homologous to a phospholipase C catalytic domain⁴Potential co-activity when expressed or paired with another toxin suchas Axmi052 ⁵Potential co-activity when expressed or paired with anothertoxin such as Axmi051

Example 2 Discovery of Novel Pesticidal Genes from Bacillusthuringiensis

Novel pesticidal genes were identified from the strains listed in Table2 using the MiDAS approach as described in U.S. Patent Publication No.20040014091, which is herein incorporated by reference in its entirety,using the following steps:

-   -   Preparation of extrachromosomal DNA from the strain.        Extrachromosomal DNA contains a mixture of some or all of the        following: plasmids of various size; phage chromosomes; genomic        DNA fragments not separated by the purification protocol; other        uncharacterized extrachromosomal molecules.    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments.    -   Sequencing of the fragmented DNA    -   Identification of putative toxin genes via homology and/or other        computational analyses.    -   When required, sequence finishing of the gene of interest by one        of several PCR or cloning strategies (e.g. TAIL-PCR).

TABLE 2 List of novel genes isolated from Bacillus thuringiensis PercentAmino identity to Nucleotide Acid closest Closest SEQ ID SEQ ID sequencesequence Gene Strain NO: NO: in art in art axmi079 ATX12974 23 83 36.7%Cry32Da1 axmi080 ATX12974 24 84 39.9% Cry42Aa1 axmi081 ATX12974 25 85  68% Orf3, described as ‘C- terminal half of a Cry Protein’ axmi082ATX13056 26 86 47.8% Cry32Da1 axmi091 ATX13053 29 89 35.3% Cry8Ba1axmi092 ATX13053 30 90 74.4% Cry39Orf2 axmi096 ATX13007 32 92 29.6%Cry32Da1 axmi097 ATX13007 33 93 29.3% Cry32Da1 axmi098 ATX13007 34 94  56% Cry41Ab1 axmi099 ATX13007 35 95   69% axmi081   61% axmi067   60%axmi079   45% axmi075   45% Cry32Ca1 axmi100 ATX12990 36 96   77%Cry9Ca1   76% Cry9Ea1   74% axmi002   72% Cry9Bb1 axmi101 ATX13035 37 97  65% Cry7Ba1   62% axmi037   60% axmi029   58% Cry7Ab2 axmi102 ATX1305638 98   86% axmi082   65% axmi093   58% axmi059   56% Cry32Aa1 axmi103ATX13056 39 99   64% axmi082   58% Cry32Da1   56% axmi093   52% axmi059axmi104 ATX13058 40 100 19.3% axmi020 18.3% Cry21Ba1 17.1% Cry5Ba1 17.1%Cry44Aa axmi107 ATX13007 41 101   35% Vip1Aa2   34% Vip1Da1 axmi108ATX12984 42 102   90% Cry45Aa1   25% Cry23Aa1 axmi109 ATX12984 43 103  38% Cry45Aa1 axmi110 ATX12984 44 104   43% Cry32Aa1 axmi111 ATX1298445 105   34% Cry41Ab1 axmi112 ATX12987 46 106   96% Cry1Ab axmi114ATX14903 47 107 85.8% axmi043 85.8% axmi028 56.7% axmi037 57.2% Cry7Ca156.3% Cry7Ab2 axmi116 ATX12975 48 108 53.5% Cry7Ba1 53.2% axmi114 53.1%axmi028   53% axmi043 52.3% Cry7Ca1 50.3% Cry7Ab1 axmi117 ATX13029 49109 92.2% Cry22Ba1 110 48.2% Cry22Aa1 47.1% Cry22Ab1 axmi118 ATX12989 50111 25.3% axmi011 22.2% Mtx2 axmi119 ATX13029 51 112 28.8% axmi027 27.8%axmi066 27.5% Cry2Ae1 26.5% axmi076 26.2% Cry18Aa1 axmi120 ATX13034 52113   50% Cry8Aa1 53 114 49.5% axmi087 54 115 49.1% Cry8Bb1 47.8%Cry8Bc1 47.8% Cry8Da1   47% Cry8Ba1 45.8% Cry8Ca1 axmi121 ATX13034 55116 51.1% axmi013 47.9% Mtx3 axmi122 ATX13034 56 117 23.1% axmi013 22.8%Mtx2 22.1% Mtx3 21.4% axmi095 axmi123 ATX12989 57 118 26.7% Cry33Aa122.7% Cry23Aa1 21.8% Cry15Aa1   19% axmi061 axmi124 ATX9387 58 119 57.6%axmi088 29.1% axmi040 28.4% axmi049 27.1% Cry5Ab1 26.5% Cry21Ba1 26.1%Cry12Aa1 25.6% axmi074 24.4% axmi031 axmi125⁷ ATX13029 59 120 38.4%Cry10Aa1 36.7% Cry10Aa2 31.3% axmi007   31% axmi006 axmi126⁸ ATX13029 60121 82.6% axmi047 81.5% axmi084 80.9% axmi086 80.9% axmi090 80.5%axmi046 79.2% axmi048 75.3% axmi092   65% Cry4Ba1 64.1% Cry4Aa1 axmi127ATX13034 124 133   58% Cry8Da1 axmi129 ATX13015 125 134   63% Cry8Aa1axmi164 ATX22201 126 135   77% Cry8Aa1 axmi151 ATX12998 127 136   61%Cry7Ba axmi161 ATX12998 128 137   52% Cry7Ca1 axmi183 ATX14775 129 138  69% Cry9Eb1 axmi132 ATX13029 130 139   55% Cry4Ba axmi138 ATX13027 131140   47% Cry41Aa1 axmi137 ATX9387 132 141   61% Axmi075 ¹This gene isthe N-terminal portion of a split cry gene and is paired in its nativecontext with Axmi126, which represents the C-terminal portion of thesplit cry pair. These genes may act as co-toxins and may show enhanced,novel, or altered activity when co-expressed or fused. The interveningregion between Axmi125 and the downstream Axmi126 is set forth in SEQ IDNO: 122. ²This gene is the C-terminal portion of a split cry gene and ispaired in its native context with Axmi125, which represents theN-terminal portion of the split cry pair. These genes may act asco-toxins and may show enhanced, novel, or altered activity whenco-expressed or fused.

Example 3 Discovery of a Novel Toxin Gene Axmi068 from Bacillusthuringiensis Strain ATX13046

The strain encoding axmi068 was identified as follows:

-   -   Sequence information from known or suspected toxin genes was        used to generate an alignment representing conserved and        partially conserved DNA sequences within a group (family) of        toxins.    -   Polymerase chain reaction (PCR) primers were designed to        selectively amplify one or more toxin family members based on        the aligned sequence.    -   DNA isolated from bacterial strains was screened by PCR to        identify strains containing putative homologs to the target gene        family.    -   PCR products were sequenced to select a strain containing a gene        of interest.        The complete gene sequence was identified from the selected        strain via the MiDAS genomics approach (U.S. Patent Publication        No. 20040014091) as follows:    -   Preparation of extrachromosomal DNA from the strain.        Extrachromosomal DNA contains a mixture of some or all of the        following: plasmids of various size; phage chromosomes; genomic        DNA fragments not separated by the purification protocol; other        uncharacterized extrachromosomal molecules.    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments.    -   Cloning of the extrachromosomal DNA fragments into a plasmid        vector.    -   Growth and purification of the cloned of the extrachromosomal        DNA.    -   Partial sequencing of the clones.    -   Identification of putative toxin genes via homology and/or other        computational analyses.    -   When required, sequence finishing (walking) of clones containing        sequence of the putative toxin genes of interest.    -   The nucleotide sequence for axmi068 is set forth in SEQ ID NO:16        and the amino acid sequence for AXMI068 is set forth in SEQ ID        NO:76.        Gene and Protein Characteristics

Gene length, DNA base pairs: 1,791 Protein length, amino acid residues:597 Estimated protein molecular weight, Da: 66,495 Known homologs andapproximate percent identity: Cry1Id1, 71.4%

Example 4 Expression in Bacillus

The insecticidal gene disclosed herein is amplified by PCR, and the PCRproduct is cloned into the Bacillus expression vector pAX916, or anothersuitable vector, by methods well known in the art. The resultingBacillus strain, containing the vector with axmi gene is cultured on aconventional growth media, such as CYS media (10 g/l Bacto-casitone; 3g/l yeast extract; 6 g/l KH₂PO₄; 14 g/l K₂HPO₄; 0.5 mM MgSO₄; 0.05 mMMnCl₂; 0.05 mM FeSO₄), until sporulation is evident by microscopicexamination. Samples are prepared and tested for activity in bioassays.

Example 5 Construction of Synthetic Sequences

In one aspect of the invention, synthetic axmi sequences were generated.These synthetic sequences have an altered DNA sequence relative to theparent axmi sequence, and encode a protein that is collinear with theparent AXMI protein to which it corresponds, but lacks the C-terminal“crystal domain” present in many delta-endotoxin proteins. Syntheticgenes are presented in Table 3.

TABLE 3 Wildtype Gene Name Synthetic Gene Name SEQ ID NO: axmi050axmi050bv01 142 axmi050bv02 143 axmi051 axmi051bv01 144 axmi051bv02 145axmi052 axmi052bv01 146 axmi052bv02 147 axmi053 axmi053bv01 148axmi053bv02 149 axmi054 axmi054bv01 150 axmi054bv02 151 axmi055axmi055bv01 152 axmi055bv02 153 axmi056 axmi056bv01 154 axmi056bv02 155axmi057 axmi057bv01 156 axmi057bv02 157 axmi058 axmi058bv01 158axmi058bv02 159 axmi059 axmi059_1bv01 160 axmi059_1bv02 161axmi059_2bv01 162 axmi059_2bv02 163 axmi060 axmi060bv01 164 axmi060bv02165 axmi061 axmi061bv01 166 axmi061bv02 167 axmi067 axmi067bv01 168axmi067bv02 169 axmi069 axmi069bv01 170 axmi069bv02 171 axmi071axmi071bv01 172 axmi071bv02 173 axmi072 axmi072bv01 174 axmi072bv02 175axmi073 axmi073bv01 176 axmi073bv02 177 axmi074 axmi074bv01 178axmi074bv02 179 axmi075 axmi075bv01 180 axmi075bv02 181 axmi079axmi079bv01 182 axmi079bv02 183 axmi080 axmi080bv01 184 axmi080bv02 185axmi082 axmi082bv01 186 axmi082bv02 187 axmi087 axmi087_1bv01 188axmi087_1bv02 189 axmi087_2bv01 190 axmi087_2bv02 191 axmi088axmi088bv01 192 axmi088bv02 193 axmi091 axmi091bv01 194 axmi091bv02 195axmi093 axmi093bv01 196 axmi093bv02 197 axmi096 axmi096bv01 198axmi096bv02 199 axmi097 axmi097_1bv01 200 axmi097_1bv02 201axmi097_2bv01 202 axmi097_2bv02 203 axmi098 axmi098bv01 204 axmi098bv02205 axmi100 axmi100bv01 206 axmi100bv02 207 optaxmi100v01 282optaxmi100v02 283 axmi101 axmi101_1bv01 208 axmi101_1bv02 209axmi101_2bv01 210 axmi101_2bv02 211 axmi102 axmi102bv01 212 axmi102bv02213 axmi103 axmi103bv01 214 axmi103bv02 215 axmi104 axmi104bv01 216axmi104bv02 217 axmi107 axmi107bv01 218 axmi107bv02 219 axmi108axmi108bv01 220 axmi108bv02 221 axmi109 axmi109bv01 222 axmi109bv02 223axmi110 axmi110bv01 224 axmi110bv02 225 axmi111 axmi111bv01 226axmi111bv02 227 axmi112 axmi112bv01 228 axmi112bv02 229 axmi114axmi114bv01 230 axmi114bv02 231 axmi116 axmi116bv01 232 axmi116bv02 233axmi117 axmi117bv01 234 axmi117bv02 235 axmi118 axmi118bv01 236axmi118bv02 237 axmi119 axmi119bv01 238 axmi119bv02 239 axmi120axmi120_1bv01 240 axmi120_1bv02 241 axmi120_2bv01 242 axmi120_2bv02 243axmi121 axmi121bv01 244 axmi121bv02 245 axmi122 axmi122bv01 246axmi122bv02 247 axmi123 axmi123bv01 248 axmi123bv02 249 axmi124axmi124bv01 250 axmi124bv02 251 axmi125 axmi125bv01 252 axmi125bv02 253axmi127 axmi127_1bv01 254 axmi127_1bv02 255 axmi127_2bv01 256axmi127_2bv02 257 axmi129 axmi129_1bv01 258 axmi129_1bv02 259axmi129_2bv01 260 axmi129_2bv02 261 axmi137 axmi137bv01 262 axmi137bv02263 axmi138 axmi138bv01 264 axmi138bv02 265 axmi151 axmi151_1bv01 266axmi151_1bv02 267 axmi151_2bv01 268 axmi151_2bv02 269 axmi161axmi161_1bv01 270 axmi161_1bv02 271 axmi161_2bv01 272 axmi161_2bv02 273axmi164 axmi164_1bv01 274 axmi164_1bv02 275 axmi164_2bv01 276axmi164_2bv02 277 axmi183 axmi183_2bv01 278 axmi183_2bv02 279axmi183bv01 280 axmi183bv02 281

In another aspect of the invention, modified versions of synthetic genesare designed such that the resulting peptide is targeted to a plantorganelle, such as the endoplasmic reticulum or the apoplast. Peptidesequences known to result in targeting of fusion proteins to plantorganelles are known in the art. For example, the N-terminal region ofthe acid phosphatase gene from the White Lupin Lupinus albus (GenebankID GI:14276838; Miller et al. (2001) Plant Physiology 127: 594-606) isknown in the art to result in endoplasmic reticulum targeting ofheterologous proteins. If the resulting fusion protein also contains anendoplasmic retention sequence comprising the peptideN-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the “KDEL”motif (SEQ ID NO:123) at the C-terminus, the fusion protein will betargeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Example 6 Expression of axmi100 in E. coli and Bacillus

The complete ORF of axmil00 (3.45 kb which encode 1156 amino acid longprotein) was cloned into an E. coli expression vector based on pRSF1b(to give pAX5445) and Bacillus vector based on pAX916 (to give pAX5444).The resulting clones were confirmed by restriction analysis and finally,by complete sequencing of the cloned gene.

For expression in E. coli, BL21*DE3 was transformed with pAX5445. Singlecolony was inoculated in LB supplemented with kanamycin and grownovernight at 37oC. Next day, fresh medium was inoculated in duplicatewith 1% of overnight culture and grown at 37oC to logarithmic phase.Subsequently, cultures were induced with 1 mM IPTG for 3 hours at 37oCor overnight at 20oC. Each cell pellet was suspended in 50 mM sodiumcarbonate buffer, pH 10.5 supplemented with 1 mM DTT and sonicated.Analysis by SDS-PAGE detected expression of a 130 kD proteincorresponding to Axmi100.

For expression in Bacillus, Bacillus thuringiensis was transformed withpAX5444 and a single colony was grown in CYS-glu medium for 3 days tosporulation. Cell pellet was then extracted with 50 mM sodium carbonatebuffer, pH 10.5 supplemented with 1 mM DTT. Soluble fraction showedpresence of a 130 kD Axmi100 protein along with several smallermolecular weight protein bands due to processing of Axmi100.Trypsinization of Axmi100 gave 2 distinct protein bands of about 65 kDand 55 kD.

Example 7 Bioassay of Axmi100

Preparation of Samples:

Cell free extracts from cells expressing AXMI-100 were typicallyresuspended in 50 mM sodium carbonate buffer, pH 10.5, typically withinclusion of 1 mM DTT as a reducing agent. Samples with and withouttrypsin were prepared for bioassay testing.

Bioassay Methodology Overview:

24-Well tissue culture plates (Corning) were given 1 ml of multi-speciesdiet (Bio-Serv) and allowed to solidify. Once solidified, 40 μl ofprotein sample was placed on the diet surface of each well and allowedto soak in/dry at room temperature. Depending upon the experiment,either ECB egg masses, ten neonate larvae or a single neonate larvaewere placed in each well. Plates were sealed with gas-permeablemembranes (Research Products International) and incubated at 25° C. and90% RH. After five or seven days (experiment dependent), samples werescored visually compared to a buffer only or non-transformed extractcontrol.

Strong activity of AXMI-100 was observed on European Corn Borer andTobacco Budworm. Activity on black cutworm was observed at high proteinconcentrations. Some activity was also observed at high concentrationson Velvet Bean caterpillar, but the activity of both black cutworm andvelvet bean caterpillar was less pronounced and more variable than forthe other insects tested. Trypsinization of Axmi100 gave 2 distinctprotein bands of about 65 kD and 55 kD, and did not appear to berequired for activity of AXMI-100.

Example 8 Additional Assays for Pesticidal Activity

The ability of a pesticidal protein to act as a pesticide upon a pest isoften assessed in a number of ways. One way well known in the art is toperform a feeding assay. In such a feeding assay, one exposes the pestto a sample containing either compounds to be tested, or controlsamples. Often this is performed by placing the material to be tested,or a suitable dilution of such material, onto a material that the pestwill ingest, such as an artificial diet. The material to be tested maybe composed of a liquid, solid, or slurry. The material to be tested maybe placed upon the surface and then allowed to dry. Alternatively, thematerial to be tested may be mixed with a molten artificial diet, thendispensed into the assay chamber. The assay chamber may be, for example,a cup, a dish, or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson, J. L. & H. K. Preisler. 1992.Pesticide bioassays with arthropods. CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals “ArthropodManagement Tests” and “Journal of Economic Entomology” or by discussionwith members of the Entomological Society of America (ESA).

Example 9 Bioassay of Axmi079 and Axmi082

Gene Expression and Purification

-   -   The DNA regions encoding the toxin domains of Axmi079 and        Axmi082 were separately cloned into an E. coli expression vector        pMAL-C4x behind the malE gene coding for Maltose binding protein        (MBP). These in-frame fusions resulted in MBP-Axmi fusion        proteins expression in E. coli.    -   For expression in E. coli, BL21*DE3 was transformed with        individual plasmids. Single colony was inoculated in LB        supplemented with carbenicillin and glucose, and grown overnight        at 37° C. The following day, fresh medium was inoculated with 1%        of overnight culture and grown at 37° C. to logarithmic phase.        Subsequently, cultures were induced with 0.3 mM IPTG for        overnight at 20° C. Each cell pellet was suspended in 20 mM        Tris-Cl buffer, pH 7.4+200 mM NaCl+1 mM DTT+protease inhibitors        and sonicated. Analysis by SDS-PAGE confirmed expression of        fusion proteins.    -   Total cell free extracts were run over amylose column attached        to FPLC for affinity purification of MBP-axmi fusion proteins.        Bound fusion protein was eluted from the resin with 10 mM        maltose solution. Purified fusion proteins were then cleaved        with either Factor Xa or trypsin to remove the amino terminal        MBP tag from the Axmi protein. Cleavage and solubility of the        proteins was determined by SDS-PAGE.        Insect Bioassays    -   Cleaved proteins were tested in insect assays with appropriate        controls. A 5-day read of the plates showed following activities        of these proteins.

MBP-Axmi fusion Axmi protein protein cleaved with Activity on insectsAxmi079 Factor Xa, trypsin Diamondback moth Axmi082 Factor Xa, trypsinDiamondback mothAdditional Insect Bioassay Results:

Gene sample tested C. elegans VBC* DBM* SWCB* CPB* ECB* Hz* Hv* Axmi50crude 3, 3 extract Axmi52 purified, 1, 0% digested Axmi58 purified, 4,digested 100% Axmi68 crude 3, 2 extract Axmi88 purified, 1, 0% 1, 0%digested Axmi93 purified, 20% digested Axmi97 purified, 1, 0% digestedAxmi102 crude 4, 3, extract 100% 75% Axmi112 purified, 3, 0% 4, 3, 25%3, 1, 3, digested 100% 75% 0% 0% Axmi117 purified, 1, 25% digestedAxmi100 purified, 4, 4, digested 100% 100% VBC = Velvetbean caterpillarDBM = diamondback moth SWCB = Southwestern corn borer CPB = Coloradopotato beetle ECB = European corn borer Hz = Helicoverpa zea Hv =Heliothis virescens *= represented as stunt and mortality percent wherestunting is scored according to the following scale: Score Definition 0No Activity 1 Slight, non-uniform stunt 2 Non-uniform stunt 3 Uniformstunt 4 Uniform stunt with mortality (expressed as a percentage) 5Uniform stunt with 100% mortality

Example 10 Vectoring of the Pesticidal Genes of the Invention for PlantExpression

Each of the coding regions of the genes of the invention are connectedindependently with appropriate promoter and terminator sequences forexpression in plants. Such sequences are well known in the art and mayinclude the rice actin promoter or maize ubiquitin promoter forexpression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35Spromoter for expression in dicots, and the nos or PinII terminators.Techniques for producing and confirming promoter—gene—terminatorconstructs also are well known in the art.

Example 11 Transformation of the Genes of the Invention into Plant Cellsby Agrobacterium-Mediated Transformation

Ears are collected 8-12 days after pollination. Embryos are isolatedfrom the ears, and those embryos 0.8-1.5 mm in size are used fortransformation. Embryos are plated scutellum side-up on a suitableincubation media, and incubated overnight at 25° C. in the dark.However, it is not necessary per se to incubate the embryos overnight.Embryos are contacted with an Agrobacterium strain containing theappropriate vectors for Ti plasmid mediated transfer for 5-10 min, andthen plated onto co-cultivation media for 3 days (25° C. in the dark).After co-cultivation, explants are transferred to recovery period mediafor five days (at 25° C. in the dark). Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated as known in the art.The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants.

Example 12 Transformation of Maize Cells with the Pesticidal Genes ofthe Invention

Maize ears are collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size are usedfor transformation. Embryos are plated scutellum side-up on a suitableincubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol;1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1mg/mL Stock) 2,4-D), and incubated overnight at 25° C. in the dark.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to express the genes of the invention in plantcells are accelerated into plant tissue using an aerosol beamaccelerator, using conditions essentially as described in PCTPublication No. WO/0138514. After beaming, embryos are incubated for 30min on osmotic media, then placed onto incubation media overnight at 25°C. in the dark. To avoid unduly damaging beamed explants, they areincubated for at least 24 hours prior to transfer to recovery media.Embryos are then spread onto recovery period media, for 5 days, 25° C.in the dark, then transferred to a selection media. Explants areincubated in selection media for up to eight weeks, depending on thenature and characteristics of the particular selection utilized. Afterthe selection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated by methods known inthe art. The resulting shoots are allowed to root on rooting media, andthe resulting plants are transferred to nursery pots and propagated astransgenic plants.

Materials

DN62A5S Media Components per liter Source Chu'S N6 Basal 3.98 g/LPhytotechnology Labs Salt Mixture (Prod. No. C 416) Chu's N6 Vitamin 1mL/L (of 1000x Phytotechnology Labs Solution (Prod. Stock) No. C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casaminoacids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L(of 1 mg/mL Sigma D-7299) Stock)

Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N KCl, addGelrite (Sigma) to 3 g/L, and autoclave. After cooling to 50° C., add 2ml/L of a 5 mg/ml stock solution of Silver Nitrate (PhytotechnologyLabs). Recipe yields about 20 plates.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A construct comprising a heterologous promoteroperably linked to a nucleic acid encoding an polypeptide havingpesticidal activity against a lepidopteran or hemipteran pest, whereinsaid nucleic acid is a nucleotide sequence selected from the groupconsisting of: a) the nucleotide sequence of SEQ ID NO:50; and b) anucleotide sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO:111.
 2. The construct of claim 1, wherein saidnucleic acid is a synthetic sequence that has been designed forexpression in a plant.
 3. The construct of claim 2, wherein said nucleicacid SEQ ID NO:236 or
 237. 4. A vector comprising the construct ofclaim
 1. 5. The vector of claim 4, further comprising a nucleic acidmolecule encoding a heterologous polypeptide.
 6. A host cell thatcontains the construct of claim
 1. 7. The host cell of claim 6 that is abacterial host cell.
 8. The host cell of claim 6 that is a plant cell.9. A transgenic plant comprising the host cell of claim
 8. 10. Thetransgenic plant of claim 9, wherein said plant is selected from thegroup consisting of maize, sorghum, wheat, cabbage, sunflower, tomato,crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,tobacco, barley, and oilseed rape.
 11. An isolated polypeptide withpesticidal activity against a lepidopteran or hemipteran pest, whereinthe polypeptide comprises a heterologous leader sequence or a transitpeptide operably linked to a protein selected from the group consistingof: a) a protein comprising the amino acid sequence of SEQ ID NO:111;and b) the protein that is encoded by SEQ ID NO:50, 236, or
 237. 12. Acomposition comprising the polypeptide of claim
 11. 13. The compositionof claim 12, wherein said composition is selected from the groupconsisting of a powder, dust, pellet, granule, spray, emulsion, colloid,and solution.
 14. The composition of claim 12, wherein said compositionis prepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof Bacillus thuringiensis cells.
 15. The composition of claim 12,comprising from about 1% to about 99% by weight of said polypeptide. 16.A method for controlling a lepidopteran or coleopteran pest population,said method comprising contacting said population with apesticidally-effective amount of a composition comprising thepolypeptide of claim
 11. 17. A method for killing a lepidopteran orcoleopteran pest, said method comprising contacting said pest with, orfeeding to said pest, a pesticidally-effective amount of a compositioncomprising the polypeptide of claim
 11. 18. A method for producing apolypeptide with pesticidal activity, said method comprising culturingthe host cell of claim 6 under conditions in which the nucleic acidmolecule encoding the polypeptide is expressed.
 19. A plant havingstably incorporated into its genome a DNA construct comprising a nucleicacid that encodes a protein having pesticidal activity, wherein saidnucleic acid is selected from the group consisting of: a) the nucleotidesequence of SEQ ID NO:50, 236, or 237; and b) a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:111; wherein said nucleic acid is operably linked to a promoter thatdrives expression of a coding sequence in a plant cell.
 20. The plant ofclaim 19, wherein said plant is a plant cell.
 21. A transgenic seedcomprising the construct of claim
 1. 22. A method for protecting a plantfrom a pest, said method comprising introducing into said plant or cellthereof at least one expression vector comprising a nucleotide sequencethat encodes a pesticidal polypeptide, wherein said nucleotide sequenceis selected from the group consisting of: a) the nucleotide sequence ofSEQ ID NO:50, 236, or 237; and b) a nucleotide sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:111.
 23. Theconstruct claim 1, wherein said promoter is capable of directingexpression of said nucleic acid in a plant cell.